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Previous studies have consistently shown that caloric restriction (CR) decreases mitochondrial reactive oxygen species (ROS) (mitROS) generation and oxidative damage to mtDNA and mitochondrial proteins, and increases maximum longevity, although the mechanisms responsible for this are unknown. We recently found that protein restriction (PR) also produces these changes independent of energy restriction. Various facts link methionine to aging, and methionine restriction (MetR) without energy restriction increases, like CR, maximum longevity. We have thus hypothesized that MetR is responsible for the decrease in mitROS generation and oxidative stress in PR and CR. In this investigation we subjected male rats to exactly the same dietary protocol of MetR that is known to increase their longevity. We have found, for the first time, that MetR profoundly decreases mitROS production, decreases oxidative damage to mtDNA, lowers membrane unsaturation, and decreases all five markers of protein oxidation measured in rat heart and liver mitochondria. The concentration of complexes I and IV also decreases in MetR. The decrease in mitROS generation occurs in complexes I and III in liver and in complex I in heart mitochondria, and is due to an increase in efficiency of the respiratory chain in avoiding electron leak to oxygen. These changes are strikingly similar to those observed in CR and PR, suggesting that the decrease in methionine ingestion is responsible for the decrease in mitochondrial ROS production and oxidative stress, and possibly part of the decrease in aging rate, occurring during caloric restriction.
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The FASEB Journal Research Communication
Methionine restriction decreases mitochondrial oxygen
radical generation and leak as well as oxidative
damage to mitochondrial DNA and proteins
Alberto Sanz,* Pilar Caro,* Victoria Ayala,
Manuel Portero-Otin,
Reinald Pamplona,
and Gustavo Barja*
*Department of Animal Physiology-II, Complutense University, Madrid, Spain;
Department of Basic
Medical Sciences, University of Lleida, Lleida, Spain
ABSTRACT Previous studies have consistently shown
that caloric restriction (CR) decreases mitochondrial
reactive oxygen species (ROS) (mitROS) generation
and oxidative damage to mtDNA and mitochondrial
proteins, and increases maximum longevity, although
the mechanisms responsible for this are unknown. We
recently found that protein restriction (PR) also pro-
duces these changes independent of energy restriction.
Various facts link methionine to aging, and methionine
restriction (MetR) without energy restriction increases,
like CR, maximum longevity. We have thus hypothe-
sized that MetR is responsible for the decrease in
mitROS generation and oxidative stress in PR and CR.
In this investigation we subjected male rats to exactly
the same dietary protocol of MetR that is known to
increase their longevity. We have found, for the first
time, that MetR profoundly decreases mitROS produc-
tion, decreases oxidative damage to mtDNA, lowers
membrane unsaturation, and decreases all five markers
of protein oxidation measured in rat heart and liver
mitochondria. The concentration of complexes I and
IV also decreases in MetR. The decrease in mitROS
generation occurs in complexes I and III in liver and in
complex I in heart mitochondria, and is due to an
increase in efficiency of the respiratory chain in avoid-
ing electron leak to oxygen. These changes are strik-
ingly similar to those observed in CR and PR, suggest-
ing that the decrease in methionine ingestion is
responsible for the decrease in mitochondrial ROS
production and oxidative stress, and possibly part of
the decrease in aging rate, occurring during caloric
restriction.—Sanz, A., Caro, P., Ayala, V., Portero-Otin,
M., Pamplona, R., Barja, G. Methionine restriction
decreases mitochondrial oxygen radical generation and
leak as well as oxidative damage to mitochondrial DNA
and proteins. FASEB J. 20, 1064–1073 (2006)
Key Words: mitochondria methionine restriction caloric re-
striction free radicals aging DNA damage oxidative
The results of many experimental and comparative
studies are consistent with the validity of the mitochon-
drial oxygen radical theory of aging (1,2). These studies
suggest that the mitochondrial rate of generation of
ROS (mitROS generation) plays a main role. Many
investigations have shown that mitROS generation is
lower in long-lived than in short-lived animal species
and that caloric restriction (CR) consistently decreases
the rate of mitROS production (reviewed in ref 3). In
these two models, low rates of mitROS generation are
accompanied by lower levels of oxidative damage to
mitochondrial DNA (mtDNA) and proteins (2,46).
Although numerous studies have documented the
decrease in MitROS production and oxidative damage
to mitochondrial macromolecules in CR, the dietary
factor that causes these beneficial changes is unknown.
Recent studies are beginning to clarify this subject.
Thus, while lipid restriction does not change mitROS
production (7), we recently found that protein restric-
tion decreases mitROS production in rat liver (8)
independent of energy intake, although the protein
component responsible for this is not known. This
decrease occurs specifically at complex I, lowers the
percent free radical leak (FRL) in the respiratory chain,
and decreases oxidative damage to mtDNA in rat liver
(8). All these changes also occur in CR with a similar
magnitude, suggesting that restriction of protein intake
can be responsible for the well-known decreases in
mitROS production and oxidative stress that take place
in CR. On the other hand, although it has been
believed that the antiaging effect of CR is due to the
decreased intake of calories themselves rather than to
decreases in specific dietary components, recent find-
ings question this consensus (9). Moreover, classic
studies have repeatedly found that protein restriction
increases maximum longevity in rats and mice (10–13),
although the magnitude of this increases are around
half that of CR. In addition, it has been observed that
methionine restriction (MetR) without energy restric-
tion increases maximum longevity in rats and mice
Correspondence: Departamento de Fisiologı´a Animal-II,
Facultad de Ciencias Biolo´gicas, Universidad Complutense,
c/Antonio Novais-2, Madrid 28040, Spain. E-mail: gbarja@
doi: 10.1096/fj.05–5568com
1064 0892-6638/06/0020-1064 © FASEB
(14,15). Various other kinds of recent investigations
also point to a relationship between methionine and
longevity (16–19).
Taking into account all these findings, we hypothe-
sized that the restriction in methionine intake can be
the cause of the decrease in mitROS generation and
oxidative stress observed in CR and in protein restric-
tion in rodents, and so can be responsible for around
half of the increase in maximum longevity observed in
caloric restriction. To test that hypothesis, in the
present investigation we subjected male rats to exactly
the same dietary protocol of MetR that is known to
increase rat maximum longevity (14). The time of
restriction used (6 –7 wk) was the same that induces
decreases in mitROS generation in the liver of rats
subjected to both caloric restriction (20) and protein
restriction (8). In liver and heart mitochondria of these
methionine-restricted rats, we measured the rates of
mitROS generation, mitochondrial oxygen consump-
tion, FRL, the concentration of complexes I and IV, five
oxidative damage markers of protein oxidation (the
specific protein carbonyls glutamic and aminoadipic
semialdehydes, GSA and AASA), glycoxidation (car-
boxyethyl-lysine CEL and carboxymethyl-lysine CML)
and lipoxidation (malondialdehydelysine MDAL, and
CML), as well as oxidative damage to mitochondrial
DNA. Since it is known that the degree of unsaturation
of phospholipids can affect markers of protein lipoxi-
dation, the full fatty acid composition was measured in
liver and heart mitochondria of methionine-restricted
and control animals.
Animals and diets
Male Wistar rats of 250 g of body wt were obtained from
Iffa-Creddo (Lyon, France). The animals were caged individ-
ually and maintained in a 12:12 (light:dark) cycle at 22 2°C
and 50 10% relative humidity. The semipurified diets used
in this investigation were prepared by MP Biochemicals
(formerly ICN), Irvine, CA, USA. Control animals were fed ad
libitum a semipurified diet based on the American Institute of
Nutrition AIN-93G diet: 31.80% cornstarch, 31.80% sucrose,
5.00% dextrin, 8.00% corn oil, 5.00% alphacel non-nutritive
bulk, 1.12% l-arginine, 1.44% l-lysine, 0.33% l-hystidine,
1.11% l-leucine, 0.82% l-isoleucine, 0.82% l-valine, 0.82%
l-threonine, 0.18% l-tryptophan, 0.86% l-methionine, 2.70%
l-glutamic acid, 1.16% l-phenylalanine, 2.33% glycine, 0.20%
choline bitartrate, 1.00% AIN vitamin mix, and 3.50% AIN
mineral mix. The methionine-restricted rats received the
same diet except that l-methionine was 0.17% and l-glutamic
acid was 3.39%. Control animals received daily the same
amount of food that the methionine-restricted animals had
eaten as a mean during the previous day (pair feeding). Using
this diet and protocol, it has been demonstrated that methi-
onine restriction increases maximum longevity in rats (14).
After 6 –7 wk of dietary treatment the animals were sacrificed
by decapitation. The liver and heart were immediately pro-
cessed to isolate mitochondria, and liver and heart samples
were stored at 80°C for the assays of oxidative damage to
Mitochondria isolation
Rat liver and heart mitochondria were obtained from fresh
tissue. The liver was rinsed and homogenized in 60 ml of
isolation buffer (210 mM mannitol, 70 mM sucrose, 5 mM
HEPES, 1 mM EDTA, pH 7.35). The nuclei and cell debris
were removed by centrifugation at 1000 g for 10 min. Super-
natants were centrifuged at 10,000 g for 10 min and the
resulting supernatants were eliminated. The pellets were
resuspended in 40 ml of isolation buffer without EDTA and
centrifuged at 1000 g for 5 min. Mitochondria were obtained
after centrifugation of the supernatants at 10,000 g for 10
min. After each centrifugation step, any overlaying layer of fat
was eliminated. Heart mitochondria were isolated by differ-
ential centrifugation as described previously (4) except that
the centrifugations were performed at 1000 g for 10 min to
eliminate nuclei and cellular debris and at 10,000 g to pellet
mitochondria. The mitochondrial pellets were resuspended
in 1 ml of isolation buffer without EDTA. All the above
procedures were performed at 5°C. Mitochondrial protein
was measured by the Biuret method. The final mitochondrial
suspensions were maintained over ice and used immediately
for measurements of oxygen consumption and H
Mitochondrial H
The rate of mitochondrial H
production was assayed by
measuring the increase in fluorescence (excitation at 312 nM,
emission at 420 nM) due to oxidation of homovanillic acid by
in the presence of horseradish peroxidase (21). Reac
tion conditions were 0.25 mg of mitochondrial protein per
ml, 6U/ml of horseradish peroxidase, 0.1 mM homovanillic
acid, 50 U/ml of superoxide dismutase (SOD), and 2.5 mM
pyruvate/2.5 mM malate, 2.5 mM glutamate/2.5 mM malate,
or 5 mM succinate 2 M rotenone as substrates, added at
the end to start the reaction to the incubation buffer (145
mM KCl, 30 mM HEPES, 5 mM KH
, 3 mM MgCl
, 0.1
mM EGTA, 0.1% albumin, pH 7.4) at 37°C, in a total vol of
1.5 ml. Unless otherwise stated, the assays with succinate as
substrate were performed in the presence of rotenone in
order to avoid the backward flow of electrons to Complex I.
In some experiments rotenone (2 M) or antimycin A (2
M) were also included in the reaction mixture to assay
maximum rates of Complex I or Complex III H
tion. Duplicated samples were incubated for 15 min at 37°C;
the reaction was stopped by transferring the samples to a cold
bath and adding 0.5 ml of stop solution (2.0 M glycine, 2.2M
NaOH, 50 mM EDTA), and the fluorescence was read in a
LS50B Perkin-Elmer fluorometer. Known amounts of H
generated in parallel by glucose (Glc) oxidase with Glc as
substrate were used as standards. Since the SOD added in
excess converts all O
excreted by mitochondria (if any) to
, the measurements represent the total (O
) rate of mitochondrial ROS production.
Mitochondrial oxygen consumption
The oxygen consumption of liver and heart mitochondria was
measured at 37°C in a water-thermostatized incubation cham-
ber with a computer-controlled Clark-type O
electrode (Oxy
graph, Hansatech, UK) in 0.5 ml of the same incubation
buffer used for H
measurements. The substrates used
were complex I- (2.5 mM pyruvate/2.5 mM malate or 2.5 mM
glutamate/2.5 mM malate) or complex II-linked (5 mM
succinate2 M rotenone). The assays were performed in
the absence (State 4, resting) and in the presence (State 3,
phosphorylating) of 500 M ADP.
Mitochondrial free radical leak
The H
production and O
consumption of liver and heart
mitochondria were measured in parallel in the same samples
under similar experimental conditions. This allowed the
calculation of the fraction of electrons out of sequence, which
reduce O
to ROS at the respiratory chain (the percent free
radical leak, FRL) instead of reaching cytochrome oxidase to
reduce O
to water. Since two electrons are needed to reduce
1 mol of O
to H
whereas four electrons are transferred in
the reduction of 1 mol of O
to water, the percent free radical
leak was calculated as the rate of H
production divided by
twice the rate of O
consumption, and the result was multi
plied by 100.
Measurement of 8-oxodG in mtDNA
Mitochondrial DNA (mtDNA) was isolated by the method of
Latorre et al. (22) adapted to mammals (23). The isolated
nuclear and mitochondrial DNAs were digested to de-
oxynucleoside level by incubation at 37°C with5Uof
nuclease P1 (in 20 l of 20 mM sodium acetate, 10 mM ZnCl
15% glycerol, pH 4.8) for 30 min and1Uofalkaline
phosphatase (in 20 l of 1 M Tris-HCl, pH 8.0) for 1 h (24).
All aqueous solutions used for mtDNA isolation, digestion,
and chromatographic separation were prepared in HPLC-
grade water. Steady-state oxidative damage to mtDNA was
estimated by measuring the concentration of 8-oxodG, re-
ferred to that of the nonoxidized base (deoxyguanosine, dG).
8-oxodG and dG were analyzed by HPLC with on-line elec-
trochemical and UV detection, respectively. The nucleoside
mixture was injected into a reverse-phase Beckman Ultra-
sphere ODS column (5 m, 4.6 mM25 cm) and was eluted
with a mobile phase containing 2.5% acetonitrile and 50 mM
phosphate buffer pH 5.0. A Waters 510 pump at 1 l/min was
used. 8-oxodG was detected with an ESA Coulochem II
electrochemical coulometric detector (ESA, Inc. Bedford,
MA, USA) with a 5011 analytical cell run in the oxidative
mode (225 mV/20 nA), and dG was detected with a Bio-Rad
model 1806 UV detector at 254 nM. For quantification, peak
areas of dG standards and of three concentration calibration
pure 8-oxodG standards (Sigma) were analyzed during each
HPLC run. A comparison of areas of 8-oxodG standards
injected with and without simultaneous injection of dG
standards ensured that no oxidation of dG occurred during
the HPLC run.
GSA, AASA, CML, CEL, and MDAL measurements in
mitochondrial proteins
GSA, AASA, CML, CEL, and MDAL concentrations were
measured by GC/MS as described previously (25). Briefly,
samples containing 500 g of mitochondrial protein were
delipidated using chloroform:methanol (2:1 v/v) in the pres-
ence of 0.01% butylated hydroxytoluene, and proteins were
precipitated by adding trichloroacetic acid to 10% (v/v) final
concentration, followed by centrifugation. Protein samples
were immediately reduced by overnight incubation with 500
in 0.2 M borate buffer at pH 9.2, and containing
1 drop of hexanol as an antifoam reagent. Protein was
reprecipitated by adding 1 ml of 20% (v/v) trichloroacetic
acid, followed by centrifugation. Isotopically labeled internal
standards ([
]-lysine (CDN Isotopes, Pointe-Claire, Que
bec, Canada) were then added; [
]-CML, [
]-CEL, and
]-MDAL were prepared as described previously (26 –28);
]5-hydroxy-2-aminovaleric acid (for GSA quantification)
and [
]6-hydroxy-2-aminocaproic acid (for AASA quantifi
cation) were prepared as described previously (29). The
samples were hydrolyzed at 155°C for 30 min in 1 ml of 6 M
HCl and dried in vacuo. The N,O-trifluoroacetyl methyl ester
derivatives of the protein hydrolysates were prepared as
described previously (26). GC/MS analyses were carried out
on a Hewlett-Packard model 6890 gas chromatograph
equipped with a HP-5MS capillary column (30 m0.25
mM0.25 m) coupled to a Hewlett-Packard model 5973A
mass selective detector. The injection port was maintained at
275°C; the temperature program was 5 min at 110°C, then
2°C/min to 150°C, then 5°C/min to 240°C, then 25°C/min
to 300°C, and finally held at 300°C for 5 min. Quantification
was performed by external standardization using standard
curves constructed from mixtures of deuterated and nondeu-
terated standards. Analyses were carried out by selected
ion-monitoring GC/MS (SIM-GC/MS). The ions used were
lysine and [
]-lysine, m/z 180 and 187, respectively; 5-hy
droxy-2-aminovaleric acid and [
acid (stable derivatives of GSA), m/z 280 and 285, respectively;
6-hydroxy-2-aminocaproic acid and [
caproic acid (stable derivatives of AASA), m/z 294 and 298,
respectively; CML and [
]-CML, m/z 392 and 396, respec
tively; CEL and [
]-CEL, m/z 379 and 383, respectively; and
MDAL and [
]-MDAL, m/z 474 and 482, respectively. The
amounts of product were expressed as the molar ratio of
GSA, AASA, CML, CEL, or MDAL/mol lysine.
Mitochondrial complex I and IV
The concentration of mitochondrial complexes I and IV was
estimated using Western blot analysis. Immunodetection was
performed using a monoclonal antibody specific for the
NDUFA9 subunit of complex I and subunit I of complex IV
(1:500 /1:2,000, Molecular Probes, Invitrogen Ltd., Abing-
don, Oxon, UK). An antibody to porin (1:1,000, Molecular
Probes, Invitrogen Ltd.) as a control for total mitochondrial
mass was also used to determine the proportion of complex I
and IV referred to total mitochondrial mass. Peroxidase-
coupled secondary antibodies were used from the Tropix
chemiluminescence kit (Bedford, MA, USA). Signal quantifi-
cation and recording was performed with a CCD camera-
based system (Lumi-Imager) from Boehringer Mannheim
(Mannheim, Germany).
Fatty acid analysis
Fatty acyl groups were analyzed as described previously (25).
Tissue lipids were extracted from homogenate fractions with
chloroform:methanol (2:1, v/v) in the presence of 0.01%
(w/v) butylated hydroxytoluene. The chloroform phase was
evaporated under nitrogen and the fatty acyl groups were
transesterified by incubation in 2.5 ml of 5% (v/v) metha-
nolic HCl for 90 min at 75°C. The resulting fatty acid methyl
esters were extracted by adding 1 ml of saturated NaCl
solution and 2.5 ml of n-pentane. The n-pentane phase was
separated and evaporated under nitrogen. The residue was
dissolved in 75 l of carbon disulfide and 1 l was used for
GC/MS analysis. Separation was performed in a SP2330
capillary column (30 m0.25 mM0.20 m) in a Hewlett
Packard 6890 Series II gas chromatograph. A Hewlett Packard
5973A mass spectrometer was used as detector in the elec-
tron-impact mode. The injection port was maintained at
220°C and the detector at 250°C; the temperature program
was 2 min at 100°C, then 10°C/min to 200°C, then 5°C/min
to 240°C, and finally held at 240°C for 10 min. Identification
of fatty acyl methyl esters was made by comparison with
authentic standards and on the basis of mass spectra. Results
are expressed as mol%. The following fatty acyl indices were
also calculated: saturated fatty acids (SFA): unsaturated fatty
1066 Vol. 20 June 2006 SANZ ET AL.The FASEB Journal
acids (UFA); monounsaturated fatty acids (MUFA); polyun-
saturated fatty acids from n-3 and n-6 series (PUFAn-3 and
PUFAn-6); average chain length (ACL) [(%Total1414)
(%Total1616) (%Total1818) (%Total2020)
(%Total2222)]/100; double bond index (DBI)
[(mol% monoenoic) (2⫻⌺mol% dienoic) (3⫻⌺mol%
trienoic) (4⫻⌺mol% tetraenoic) (5⫻⌺mol% pentae-
noic) (6⫻⌺mol% hexaenoic)], and peroxidizability index
(PI) [(0.025⫻⌺mol% monoenoic) (mol% dienoic)
(2⫻⌺mol% trienoic) (4⫻⌺mol% tetraenoic)
(6⫻⌺mol% pentaenoic) (8⫻⌺mol% hexaenoic)].
Data were analyzed by Student’s t tests. The minimum con-
centration of statistical significance was set at P 0.05 in all
the analyses.
Methionine restriction significantly decreased the rate
of H
production of heart mitochondria with pyru
vate/malate as substrates to values 64% those of control
animals (Fig. 1, upper panel). With succinate as sub-
strate no significant differences between control and
MetR heart mitochondria were observed (Fig. 1, upper
panel). However, when the experiments with succinate
as substrate were repeated in the absence of rotenone,
the rate of H
generation of the MetR group was
significantly lower than that of controls.
In liver mitochondria, methionine restriction also
significantly decreased the rate of H
generation to
46% and 43% of control values with pyruvate/malate
and with glutamate/malate as substrates, respectively
(Fig. 1, lower panel). In the presence of succinate,
hepatic mitochondrial H
production also was signif
icantly lower in MetR than in control mitochondria.
Maximum rates of H
generation were assayed
using appropriate combinations of substrates and in-
hibitors of the respiratory chain (Table 1). The rate of
production of heart mitochondria was signifi
cantly decreased by methionine restriction in the pres-
ence of pyruvate/malaterotenone (full reduction of
complex I) but not in the presence of succinate
antimycin A (full reduction of complex III). In the case
of liver mitochondria, no significant differences in
generation between control and MetR groups
were observed with pyruvate/malaterotenone, gluta-
mate/malaterotenone or succinateantimycin A
(Table 1).
The rate of O
consumption of heart mitochondria
was significantly increased by MetR with pyruvate/
malate in state 3 but not in state 4 (Table 2). In the
presence of succinate, nonsignificant trends to increase
heart mitochondrial O
consumption were found that
showed marginal significance (P 0.07 in state 4 and
P 0.08 in state 3).
In liver mitochondria, the rate of O
was significantly increased by MetR with glutamate/
malate and with succinate in both state 4 and state 3, as
well as with pyruvate/malate in state 3 (Table 2). Only
with pyruvate/malate in state 4 did the increase in O
consumption fail to reach statistical significance.
The percentage of electrons in the respiratory chain
directed to ROS generation (the free radical leak, FRL)
in heart mitochondria was significantly decreased by
MetR with pyruvate/malate (to 43% of controls; Fig. 2)
but not with succinate (results not shown). In liver
mitochondria, MetR significantly decreased the FRL
both with the complex I (pyruvate/malate and gluta-
mate/malate) and the complex II (succinate) linked
substrates used to values 28 –39% those of controls
(Fig. 2).
Methionine restriction significantly decreased the
concentration of complex I and complex IV in both
heart and liver mitochondria (Fig. 3).
Figure 1. Rates of H
production of heart and liver
mitochondria from control and methionine-restricted rats.
PYR pyruvate/malate; SUCC succinate; SUCC-ROT
succinate without rotenone; GLU glutamate/malate;
MetR methionine-restricted. Control values in heart mito-
chondria: 0.25 0.03 (PYR); 0.77 0.20 (SUCC); 7.24
1.00 (SUCC-ROT). Control values in liver mitochondria:
0.13 0.03 (PYR); 0.93 0.17 (GLU); 0.460 0.04 (SUCC).
Values are means se from 6 8 different animals. Asterisks
represent statistically significant differences between control
and methionine-restricted animals (*P0.05; **P0.01).
The levels of the oxidative damage marker 8-oxodG
in mtDNA were significantly decreased by MetR to 48%
of controls in liver and to 65% of controls in heart
(Fig. 4).
All the protein markers of oxidative, glycoxidative,
and lipoxidative damage were significantly decreased
by MetR in both liver and heart mitochondria (Table
3). In heart mitochondrial proteins GSA, AASA, CEL,
and CML were decreased to 67–82% of control values,
whereas in the case of MDAL the values found in MetR
were 50% those of controls (Table 3). In liver mito-
chondria, GSA, CEL, and CML were decreased by MetR
to 70 85% of controls whereas AASA and MDAL were
lowered to 68% and 63% of controls, respectively.
Methionine restriction altered the fatty acid compo-
sition of liver (Table 4) and heart (Table 5) mitochon-
dria, so that the total number of double bonds (DBI)
was significantly decreased in both cases. In both kinds
of mitochondria, the fatty acids mainly responsible for
the decrease in DBI were the same. Thus, methionine
restriction significantly increased the fatty acids with no
or a small number of double bonds 18:0, 18:1n-9, and
18:2n-6 whereas it significantly decreased the main
highly unsaturated ones 20:4n-6 and 22:6n-3 (Tables 4
and 5).
Figure 2. Free radical leak (%) of heart and liver mitochon-
dria from control and methionine-restricted rats. The FRL is
the percentage of the total electron electron flow in the
respiratory chain directed to oxygen radical generation (see
Materials and Methods). PYR, pyruvate/malate; SUCC, succi-
nate; GLU, glutamate/malate; MetR, methionine-restricted.
Values are means se from 7– 8 different animals. Asterisks
represent statistically significant differences between control
and methionine-restricted animals (*P0.05; **P0.01).
Figure 3. Concentration of protein complexes I and IV in
heart and liver mitochondria from control and methionine-
restricted rats. Values are means se from 4 –5 different
animals. Asterisks represent statistically significant differences
between control and methionine-restricted animals
TABLE 2. Oxygen consumption of heart and liver mitochondria
from control and methionine-restricted rats
Control Methionine restricted
Heart mitochondria:
Pyr (E4) 26.2 4.8 33.75.0
Pyr (E3) 123 16 185 3*
Succ (E4) 86 18 125 15
Succ (E3) 137 30 218 48
Liver mitochondria:
Pyr (E4) 5 1.2 6 1.0
Pyr (E3) 15 1.5 20 2.3*
Glu (E4) 7 1.2 10 1.2*
Glu (E3) 59 7.4 93 6.8**
Succ (E4) 17 2.0 30 2.9**
Succ (E3) 88 10.2 129 9.4**
Pyr, pyruvate/malate; Glu, Glutamate/malate; Succ, succinate;
E4, state 4 (substrate alone); E3, state 3 (substrateADP). Values are
means se from 7–8 different animals and are expressed in
nanomoles of O
/min. mg mitochondrial protein. Asterisks repre
sent statistically significant differences between control and methi-
onine-restricted groups (*P0.05; **P0.01);
TABLE 1. Maximum H
production in the presence of
respiratory chain inhibitors in heart and liver mitochondria from
control and methionine-restricted rats
Control Methionine restricted
PyrRot 3.53 0.32 2.73 0.17*
SuccAA 23.17 3.40 19.54 2.59
PyrRot 0.56 0.07 0.69 0.06
GluRot 0.82 0.09 0.74 0.08
SuccAA 3.21 0.33 3.39 0.34
Pyr, pyruvate/malate; Succ, succinate; Rot, rotenone; AA, anti
mycin A. Values are means se from 7– 8 different animals and are
expressed in mol of H
/min. mg mitochondrial protein.
*(P0.05 between control and methionine-restricted groups).
1068 Vol. 20 June 2006 SANZ ET AL.The FASEB Journal
In this work it is shown for the first time that methio-
nine restriction decreases mitochondrial ROS genera-
tion and oxidative damage to mitochondrial DNA and
proteins. This strongly suggests that the decrease in
methionine intake is the cause of these effects consis-
tently found in many investigations on caloric restric-
tion in rodents.
Caloric restriction is the better known experimental
manipulation that decreases aging rate, but its mecha-
nisms of action are unknown. Many studies have con-
sistently shown that CR decreases mitROS generation
(3), suggesting this can be one of the main mecha-
nisms. But the dietary component responsible for this
change was unknown. A recent study from our labora-
tory found that protein restriction (PR) without strong
caloric restriction also decreases mitROS generation at
complex I, lowers FRL, and decreases 8-oxodG in
mtDNA (8) in rat liver mitochondria in exactly the
same quantitative and qualitative way as CR, whereas
neither lipid restriction (7) nor carbohydrate restric-
tion (unpublished results) modify the rate of mitROS
production. Looking for the protein component re-
sponsible for these effects, we focused on methionine
in the present investigation since various sources of
data currently relate methionine to aging. First, it is
known that MetR without energy restriction increases
maximum life span in rats and mice (14,15), similar to
what was found in PR (10 –13) although the PR effect
on life span is smaller than that of CR. Recent investi-
gations suggest that the increase in maximum longevity
induced by MetR always occurs irrespective of the
particular rat strain selected (30). Second, it has been
found that the protein methionine content is inversely
Figure 4. Oxidative damage to mitochondrial DNA (8-
oxodG) in heart and liver from control and methionine-
restricted rats. MetR, methionine-restricted. Values are
means se from 6–7 different animals. Asterisks represent
statistically significant differences between control and methi-
onine-restricted animals (**P0.01).
TABLE 3. Protein markers of oxidative, glycoxidative, and lipoxidative damage in liver and heart mitochondria from control and
methionine-restricted rats
Liver mitochondria Heart mitochondria
Control MetR Control MetR
GSA 4193 96 3168 137*** 5004 121 3950 131***
AASA 137 793 2*** 212 13 143 23*
CEL 369 8 258 10*** 572 22 409 25**
CML 1201 17 1021 30*** 1768 35 1441 43***
MDAL 387 14 245 8*** 452 17 228 14***
GSA, glutamic semialdehyde; AASA, aminoadipic semialdehyde; CEL, carboxyethyl-lysine; CML, carboxymethyl-lysine; MDAL, malondi
aldehyde-lysine. MetR, methionine-restricted. Values are means se from 5–7 different animals and are expressed as mol/mol lysine. Asterisks
represent statistically significant differences between control and methionine-restricted groups (*P0.05; **P 0.01; ***P0.001).
TABLE 4. Fatty acid analysis of liver mitochondria from control
and methionine-restricted rats
Liver mitochondria
Control Methionine restriction P
14:0 0.23 0.003 0.21 0.01 0.224
16:0 16.11 0.31 15.04 0.24 0.022
16:1n-7 0.75 0.09 0.48 0.04 0.022
18:0 18.14 0.22 20.48 0.31 0.000
18:1n-9 7.13 0.40 8.73 0.57 0.064
18:2n-6 20.20 0.49 21.93 0.46 0.031
18:3n-3 0.28 0.01 0.26 0.01 0.368
20:3n-6 0.42 0.07 0.30 0.03 0.139
20:4n-6 28.05 0.44 25.23 0.29 0.000
22:4n-6 1.08 0.12 1.01 0.07 0.623
22:5n-6 0.55 0.04 0.46 0.03 0.120
22:5n-3 0.64 0.04 0.56 0.02 0.124
22:6n-3 6.36 0.16 5.25 0.11 0.000
ACL 18.56 0.01 18.48 0.01 0.000
SFA 34.48 0.13 35.73 0.28 0.006
UFA 65.51 0.13 64.26 0.28 0.006
MUFA 7.88 0.37 9.22 0.57 0.109
PUFA 57.62 0.39 55.03 0.57 0.007
PUFAn-3 7.29 0.17 6.08 0.11 0.000
PUFAn-6 50.32 0.29 48.94 0.61 0.107
DBI 211.17 1.37 196.48 1.14 0.000
PI 196.48 1.94 176.54 1.64 0.000
ACL, acyl chain length; SFA, saturated fatty acids; UFA, unsat
urated fatty acids; MUFA, monounsaturated fatty acids; PUFA, poly-
unsaturated fatty acids; DBI, double bond index; PI, poliunsaturation
index. Values are means se from 5–7 different animals and are
expressed as mol%.
related to maximum life span in mammals (16), which
makes sense since methionine is among the protein
amino acids most susceptible to oxidation by ROS (17).
Third, methionine dietary supplementation damages
many vital organs (like cardiovascular system and liver)
and increases oxidative stress (31,32), and similar neg-
ative effects have been found in rats fed high protein
diets (31,33). Fourth, knocking out methionine sulfox-
ide reductase-A (an enzyme that reduces methionine
sulfoxide back to methionine through a thioredoxin-
dependent reaction) lowers maximum longevity and
increases protein carbonyls in mice (17), whereas over-
expression of this enzyme in Drosophila increases its life
span and delays aging (18). Fifth, overexpression of
thioredoxin increases longevity in mice (19). Sixth,
long-lived mutant Ames dwarf mice seem to have
altered methionine metabolism (34).
In agreement with our hypothesis, we found in this
investigation that MetR, using the same dietary proto-
col that increases maximun longevity in rats (14), also
decreases mitROS production, FRL, and oxidative dam-
age to mtDNA, similar to what has been observed in PR
and CR (3,4,20). The decrease in mitROS production
occurred in both liver and heart mitochondria and was
quantitatively strong. In the case of heart mitochondria,
the decrease in ROS generation occurred only at
complex I since it occurred with pyruvate/malate but
not with succinate (rotenone) as substrate, and it also
took place when the assay with succinate was performed
in the absence of rotenone (which allows backward flow
of electrons from succinate to the complex I ROS
generator). In heart mitochondria it has been found
that the decrease in ROS generation in CR also occurs
exclusively at complex I (4). In the case of liver
mitochondria, MetR decreased ROS generation both at
complex I and complex III, since the decreases oc-
curred with the complex II-linked substrate (succinate)
as well as with the complex I-linked ones (pyruvate/
malate and glutamate/malate). In 40% CR, decreases
in ROS generation have been detected only at complex
I in rat liver mitochondria (20).
Concerning the mechanism responsible for the de-
crease in mitROS production during MetR, there are
various possibilities. A simple mechanism can be a
decrease in the concentration of the respiratory com-
plex/es responsible for ROS generation. This has been
described in relation to aging in comparisons between
rats and pigeons, in which the lower rate of mitROS
production of the long-lived species (the pigeon) has
been attributed to its lower complex I content (35).
This could also be a mechanism during food restric-
tion, since variations in the levels of mitochondrial
protein complexes have been described as a result of
CR (36). To test this, we measured the concentration of
complex I and IV in control and MetR animals. Com-
plex I contains the ROS generator site mainly related to
aging, whereas complex IV is known not to produce
ROS and was also measured as a control. Similar results
were found in both heart and liver mitochondria. MetR
decreased the concentration of complexes I and IV.
This strong decrease in complex I content in MetR, as
in the pigeon vs. rat comparison, can contribute to the
decrease observed in mitROS production.
Even if the decrease in complex I helps to decrease
ROS generation, it cannot be the only explanation of
this change for various reasons. First, while ROS pro-
duction with substrate alone decreases in MetR, when
maximum rates of ROS production were assayed with
appropriate combinations of substrate plus inhibitor
(pyruvate/malate plus rotenone and succinate plus
antimycin A), in almost every case the difference in
ROS generation between control and MetR mitochon-
dria disappeared. With substrate alone the respiratory
chain is only partially reduced, so that ROS production
depends both on the amount of ROS generators
present as well as on the degree of electronic reduction
of these generators (the higher their degree of reduc-
tion, the higher will be their rate of ROS production).
But in the presence of the inhibitors, the ROS genera-
tors are fully reduced, so that the degree of reduction
of the generators is no longer a variable. If the decrease
in ROS production observed in MetR were only due to
decreases in the concentration of ROS generators, a
lower ROS production would be observed in the pres-
ence of those substrateinhibitor combinations in
MetR than in the controls. Since this was not the case,
it seems that the degree of reduction of the ROS
generators is involved in the decreases in ROS produc-
tion observed in MetR with substrate alone.
That intrinsic changes at the concentration of the
ROS generators occur in MetR is also indicated by the
TABLE 5. Fatty acid analysis of heart mitochondria from control
and methionine-restricted rats
Heart mitochondria
Control Methionine restriction P
14:0 0.21 0.01 0.18 0.002 0.037
16:0 11.77 0.06 11.05 0.11 0.001
16:1n-7 0.29 0.03 0.20 0.02 0.063
18:0 23.30 0.30 24.59 0.36 0.029
18:1n-9 6.10 0.03 7.24 0.22 0.001
18:2n-6 16.69 0.42 18.37 0.19 0.014
20:4n-6 27.88 0.14 26.18 0.40 0.003
22:4n-6 2.85 0.07 2.15 0.15 0.003
22:5n-6 1.31 0.08 1.29 0.04 0.890
22:5n-3 1.43 0.04 1.32 0.02 0.064
22:6n-3 8.11 0.05 7.39 0.22 0.009
ACL 18.85 0.007 18.77 0.006 0.000
SFA 35.29 0.30 35.83 0.35 0.283
UFA 64.70 0.30 64.16 0.35 0.283
MUFA 6.39 0.06 7.44 0.24 0.002
PUFA 58.30 0.33 56.71 0.14 0.005
PUFAn-3 9.55 0.07 8.71 0.24 0.009
PUFAn-6 48.75 0.38 48.00 0.12 0.140
DBI 225.21 0.74 214.97 1.08 0.000
PI 221.26 0.93 206.74 1.24 0.000
ACL, acyl chain length; SFA, saturated fatty acids; UFA, unsat
urated fatty acids; MUFA, monounsaturated fatty acids; PUFA, poly-
unsaturated fatty acids; DBI, double bond index; PI, poliunsaturation
index. Values are means se from 5–7 different animals and are
expressed as mol%.
1070 Vol. 20 June 2006 SANZ ET AL.The FASEB Journal
strong decrease in percent free radical leak (FRL)
observed in MetR. MetR mitochondria not only pro-
duce less ROS per unit time, but also produce less ROS
per unit elecron flow in the respiratory chain, similar to
what has been observed in CR (4,20) and PR (8) rats as
well as in long-lived vs. short-lived animal species (36).
In all these models of slow aging, the mitochondria are
more efficient since they have a lower rate of ROS
generation without the need to decrease oxygen con-
sumption (and thus energy production), an interesting
capability. In the case of MetR, not only was the
consumption of oxygen not decreased, but it was even
increased, providing more clues concerning the mech-
anisms of decrease in ROS generation. Contrary to
intuitive thinking, when in the same individual the rate
of mitochondrial O
consumption increases it tends to
decrease (instead of increase) ROS generation due to
two different effects. On the one hand, the degree of
reduction of the respiratory chain decreases, which
tends to decrease ROS generation. On the other hand,
the increased oxygen consumption locally lowers the
, and this lowers ROS production since the K
the ROS generators (which have low affinity for O
falls within the range of physiological tissue pO
, mean
ing that ROS production is pO
dependent at physio
logical tissue oxygen partial pressures. For these two
reasons, the increase in oxygen consumption of the
MetR mitochondria helps lower their rate of ROS
production while decreasing their FRL (increase in
efficiency in avoiding ROS generation).
Another possibility is that the decrease in ROS pro-
duction is related to a mild uncoupling induced by the
MetR protocol. This is suggested by the general trend
to increased oxygen consumption of the MetR mito-
chondria, which suggests that the proton gradient was
lowered by MetR. This would be relevant since it is
known that high proton gradients raise ROS produc-
tion at least at complex III. In any case, such changes in
oxygen consumption do not occur in 40% CR and 40%
PR (4,8,20). The reason for this difference could be the
particular MetR protocol used in our investigation:
methionine was restricted by 80% instead of 40% (as in
the previous CR and PR studies), and it was substituted
for glutamate in the diet. More studies are needed to
ascertain whether the decrease in ROS production still
occurs and the increases in oxygen consumption disap-
pear when MetR is performed at 40% and without
substituting it for dietary glutamate.
Another mechanism can also be involved in the
decrease in mitROS production in MetR. It has been
reported that addition of oxidized glutathione to mito-
chondrial complex I increases its rate of superoxide
radical generation (37), and the same seems to occur
after addition of homocysteine to rat heart mitochon-
dria (38), suggesting that MitROS production can be
regulated by thiol agents. Toxic effects of dietary me-
thionine have been related to its conversion to and
increase in homocysteine levels (31,39). Homocysteine
has a free thiol group that can react with protein thiols,
leading to protein mixed disulfides. Thus, MetR could
decrease ROS generation through decreases in homo-
cysteine, which would decrease thyolization of complex
I. Homocysteine levels also increase with age in humans
and represent a risk factor for aging and free radical-
associated degenerative diseases (40).
Concerning oxidative damage, in this investigation it
was found that MetR decreases 8-oxodG levels in
mtDNA in both liver and heart. This also occurs in the
same rat organs after both CR (4,20) and PR (8). This
further supports the idea that the decrease in methio-
nine ingestion is responsible for the decrease in mito-
chondrial oxidative stress observed in CR and PR. The
decrease in 8-oxodG in MetR also agrees with the lower
mitROS generation observed in this dietary manipula-
tion, similar to what has been found in CR (4,20) and
PR (8). Similar reasoning can be applied to the strong
decreases in all of the five different markers of oxida-
tive damage to mitochondrial proteins measured in this
study. Decreases in GSA, MDAL, CML, and CEL have
been observed in heart mitochondrial proteins of CR
rats (5,6). Of these protein markers, some of them
(CML and MDAL) are dependent on lipid peroxida-
tion. Since lipid peroxidation increases strongly as a
function of the number of double bonds per fatty acid,
we measured the full fatty acid composition of heart
and liver mitochondria. We found that in both tissues
MetR significantly decreases the total number of dou-
ble bonds. Thus, part of the decrease in CML and
MDAL can be secondary to this decrease in the number
of fatty acid double bonds. But this cannot be the full
explanation because other protein markers, not depen-
dent on lipid peroxidation (GSA, AASA and CEL),
were also decreased by MetR, and because the quanti-
tative decrease in double bonds was relatively small
compared to that in MDAL and CML. Thus, it seems
that the decrease in mitROS generation induced by
MetR leads to a generalized decrease in oxidation,
glycoxidation, and lipoxidation of mitochondrial pro-
Concerning the particular fatty acid modified by
MetR, the most important changes observed (quantita-
tively) were the same in both tissues. MetR decreased
the highly unsaturated fatty acids 20:4n-6 and 22:6n-3,
and substituted them for the much less unsaturated
18:2n-6, 18:1n-9, and 18:0. It is striking that this is
essentially the kind of difference that has been ob-
served when comparing animals with different longevi-
ties. Long-lived animals have less unsaturated mem-
branes in tissues and mitochondria than short-lived
ones, mainly due to a decrease in 22:6n-3 and 20:4n-6
and to an increase in 18:2n-6, leading to a lower lipid
peroxidation and lipoxidation-dependent damage to
macromolecules (41).
In summary, it is shown here for the first time that
methionine restriction, similar to both caloric and
protein restriction, decreases mitochondrial ROS gen-
eration, increases the efficiency of the respiratory chain
in avoiding electron leak to oxygen, and lowers steady-
state oxidative damage to mitochondrial DNA and
proteins. This suggests that the decrease in methionine
ingestion can be the single molecular component re-
sponsible for the decrease in mitochondrial ROS gen-
eration and oxidative stress that occurs during caloric
restriction, and thus for part of the decrease in aging
rate elicited by this dietary manipulation, although a
role for other dietary amino acids cannot be discarded
without further investigation.
This study was supported in part by ID grants from the
Spanish Ministry of Science and Technology (BFI2003–
01287), the Generalitat of Catalunya (Departament
d’Universitats, Recerca, i Societat de la Informacio´, DURSI,
ref. 2005SGR00101), and the Spanish Ministry of Health (FIS
02–0891, 04 0355, and 05–2241) to R.P., from Fundacio la
Caixa to R.P. and M.P.O, and from the Spanish Ministry of
Science and Education (BFU2005– 02584) to G.B. We thank
David Argiles for excellent technical assistance. P. Caro and
A. Sanz received a predoctoral fellowship from the Ministry of
Science and Technology and from the Complutense Univer-
sity, respectively.
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Received for publication December 12, 2005.
Accepted for publication January 24, 2006.
... The restriction in dietary Met has been reported to decrease the concentrations of mitochondrial ETC, and reduce mitochondrial ROS generation in rodents, suggesting a regulatory effect of Met on mitochondrial ETC [40,41]. It has also been reported that dietary Met restriction decreases mitochondrial ROS generation primarily via inhibiting complex I activity and ROS generation rather than augmenting antioxidative capacity, thereby ameliorating oxidative damage to hepatic mitochondrial DNA and proteins [42]. ...
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Ferroptosis, a type of iron-dependent necrotic cell death, is triggered by the accumulation of excessive lipid peroxides in cells. Glutathione (GSH), a tripeptide redox molecule that contains a cysteine (Cys) unit in the center, plays a pivotal role in protection against ferroptosis. When the transsulfuration pathway is activated, the sulfur atom of methionine (Met) is utilized to generate Cys, which can then suppress Cys-starvation-induced ferroptosis. In the current study, we cultured HeLa cells in Met- and/or cystine (an oxidized Cys dimer)- deprived medium and investigated the roles of Met in ferroptosis execution. The results indicate that, in the absence of cystine or Met, ferroptosis or cell cycle arrest, respectively, occurred. Contrary to our expectations, however, the simultaneous deprivation of both Met and cystine failed to induce ferroptosis, although the intracellular levels of Cys and GSH were maintained at low levels. Supplementation with S-adenosylmethionine (SAM), a methyl group donor that is produced during the metabolism of Met, caused the cell cycle progression to resume and lipid peroxidation and the subsequent induction of ferroptosis was also restored under conditions of Met/cystine double deprivation. DNA methylation appeared to be involved in the resumption in the SAM-mediated cell cycle because its downstream metabolite S-adenosylhomocysteine failed to cause either cell cycle progression or ferroptosis to be induced. Taken together, our results suggest that elevated lipid peroxidation products that are produced during cell cycle progression are involved in the execution of ferroptosis under conditions of Cys starvation.
... Furthermore, in the case of the impaired flow of electrons in the ETC, as well as in the presence of excessive ETC substrate supply and electron overload without the dissipation of ∆ψ m by the ATP synthase, the increased production of ROS can occur [19,40]. Constantly increased levels of ROS can negatively affect mitochondrial proteins involved in OXPHOS and mtDNA, hence contributing to mitochondrial dysfunction, electron leakage from the ETC, and further ROS production and oxidative damage [41,42]. Of note, mtDNA is extremely sensitive to oxidative injury due to a lack of all necessary mtDNA repair enzymes, the absence of histones, and the proximity to the inner mitochondrial membrane, which is the main source of ROS [8]. ...
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One of the major mechanisms of drug-induced liver injury includes mitochondrial perturbation and dysfunction. This is not a surprise, given that mitochondria are essential organelles in most cells, which are responsible for energy homeostasis and the regulation of cellular metabolism. Drug-induced mitochondrial dysfunction can be influenced by various factors and conditions, such as genetic predisposition, the presence of metabolic disorders and obesity, viral infections, as well as drugs. Despite the fact that many methods have been developed for studying mitochondrial function, there is still a need for advanced and integrative models and approaches more closely resembling liver physiology, which would take into account predisposing factors. This could reduce the costs of drug development by the early prediction of potential mitochondrial toxicity during pre-clinical tests and, especially, prevent serious complications observed in clinical settings.
... Interestingly, literatures suggested that the therapeutic intervention of methionine restriction and cysteine supplementation are attributable to their antioxidant capability against oxidative stress during aging. Rats fed with methionine restriction diet showed a reduction in reactive oxygen species (ROS) generation and protected mitochondrial DNA from ROS damage in liver, brain, heart, and kidney [30]. Cysteine supplementation could ameliorate insulin resistance and age-related degenerations by improving skeletal muscle functions and reducing oxidative stress [27,29]. ...
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Background Gestational diabetes mellitus (GDM) is defined as impaired glucose tolerance in pregnancy and without a history of diabetes mellitus. While there are limited metabolomic studies involving advanced maternal age in China, we aim to investigate the metabolomic profiling of plasma and urine in pregnancies complicated with GDM aged at 35–40 years at early and late gestation. Methods Twenty normal and 20 GDM pregnant participants (≥ 35 years old) were enlisted from the Complex Lipids in Mothers and Babies (CLIMB) study. Maternal plasma and urine collected at the first and third trimester were detected using gas chromatography-mass spectrometry (GC-MS). Results One hundred sixty-five metabolites and 192 metabolites were found in plasma and urine respectively. Urine metabolomic profiles were incapable to distinguish GDM from controls, in comparison, there were 14 and 39 significantly different plasma metabolites between the two groups in first and third trimester respectively. Especially, by integrating seven metabolites including cysteine, malonic acid, alanine, 11,14-eicosadienoic acid, stearic acid, arachidic acid, and 2-methyloctadecanoic acid using multivariant receiver operating characteristic models, we were capable of discriminating GDM from normal pregnancies with an area under curve of 0.928 at first trimester. Conclusion This study explores metabolomic profiles between GDM and normal pregnancies at the age of 35–40 years longitudinally. Several compounds have the potential to be biomarkers to predict GDM with advanced maternal age. Moreover, the discordant metabolome profiles between the two groups could be useful to understand the etiology of GDM with advanced maternal age.
... The dietary methionine restriction rescued cytochrome c oxidase dysfunction in KO mice but did not significantly influence respiratory flux in WT mice. In agreement, most [70e73] but not [74] all studies evaluating the impact of dietary methionine restriction on liver mitochondrial respiration report no change in WT mice. A methioninerestricted diet has been shown to reduce hepatic cytosolic and nuclear protein synthesis [75,76]. ...
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Objective One-carbon metabolism is routinely dysregulated in nonalcoholic fatty liver disease. This includes decreased glycine N-methyltransferase (GNMT), a critical regulator of s-adenosylmethionine (SAM). Deletion of GNMT in mice increases SAM and promotes liver steatosis. Lower liver oxidative metabolism as indicated by a decline in gluconeogenesis, citric acid cycle flux, and oxidative phosphorylation contributes to liver steatosis in GNMT-null mice, however, the extent to which this phenotype is mediated by higher SAM remains unclear. Here, we determined the SAM-dependent impairment in liver oxidative metabolism by loss of GNMT. Methods GNMT knockout (KO) mice were fed a methionine-restricted diet to prevent increased SAM. ²H/¹³C metabolic flux analysis was performed in conscious, unrestrained mice to quantify liver nutrient fluxes. Metabolomics and high-resolution respirometry was used to quantify liver nutrient pool sizes and mitochondrial oxidative phosphorylation, respectively. Folic acid-supplemented and serine/glycine-deficient diets were used independently to further define the metabolic implications of perturbed one-carbon donor availability. Results Dietary methionine restriction prevented a 75-fold increase in SAM and 53% rise in triacylglycerides in livers of KO mice. Dietary methionine restriction increased gluconeogenesis independent of genotype and restored cytochrome c oxidase respiratory function in KO mice. Citric acid cycle fluxes remained lower in KO mice irrespective of diet. Restricting dietary methionine abrogated markers of increased lipogenesis and folate cycle dysfunction in KO mice. Conclusion The impaired liver oxidative metabolism following loss of GNMT is both dependent and independent of greater SAM availability. Lower in vivo citric acid cycle flux is independent of increased SAM. In contrast, gluconeogenesis and oxidative phosphorylation are negatively regulated by excess SAM. Lipid accumulation in livers of mice lacking GNMT is also linked to the higher SAM.
... Methionine restriction (MR) is proposed to reproduce some of the metabolic consequences of CR. For example, MR, like CR, leads to lower rates of mitochondrial reactive oxygen species (ROS) production and oxidative damage on mitochondrial DNA (mtDNA) in mice [107]. Suppression of the growth hormone (GH)/IGF somatotropic axis has been reported in rodents upon MR [83,104], leading to in vivo insulin sensitivity, reduced hepatic lipogenesis, and white adipose tissue lipid remodeling. ...
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One-carbon metabolism (OCM) is a network of biochemical reactions delivering one-carbon units to various biosynthetic pathways. The folate cycle and methionine cycle are the two key modules of this network that regulate purine and thymidine synthesis, amino acid homeostasis, and epigenetic mechanisms. Intersection with the transsulfuration pathway supports glutathione production and regulation of the cellular redox state. Dietary intake of micronutrients, such as folates and amino acids, directly contributes to OCM, thereby adapting the cellular metabolic state to environmental inputs. The contribution of OCM to cellular proliferation during development and in adult proliferative tissues is well established. Nevertheless, accumulating evidence reveals the pivotal role of OCM in cellular homeostasis of non-proliferative tissues and in coordination of signaling cascades that regulate energy homeostasis and longevity. In this review, we summarize the current knowledge on OCM and related pathways and discuss how this metabolic network may impact longevity and neurodegeneration across species.
... The endotoxin binds to specific receptors to inhibit enzyme dehydrogenase activity in the oxidative respiration chain, blocking energy production and impairing the MRC, which not only affect antioxidant enzyme activity but also partially block the flow of electrons, reducing the production of oxygen and leading to the accumulation of superoxide anion production within respiratory complexes I and III [63]. The resulting imbalance of ROS can lead to further damage of OXPHOS proteins and mtDNA [64][65][66]. Thus, mitochondrial dysfunction is aggravated, and activation of programmed cell death pathways is promoted. ...
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In addition to playing a pivotal role in cellular energetics and biosynthesis, mitochondrial components are key operators in the regulation of cell death. In addition to apoptosis, necrosis is a highly relevant form of programmed liver cell death. Differential activation of specific forms of programmed cell death may not only affect the outcome of liver disease but may also provide new opportunities for therapeutic intervention. This review describes the role of mitochondria in cell death and the mechanism that leads to chronic liver hepatitis and liver cirrhosis. We focus on mitochondrial-driven apoptosis and current knowledge of necroptosis and discuss therapeutic strategies for targeting mitochondrial-mediated cell death in liver diseases.
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Nutriments have been deemed to impact all physiopathologic processes. Recent evidences in molecular medicine and clinical trials have demonstrated that adequate nutrition treatments are the golden criterion for extending healthspan and delaying ageing in various species such as yeast, drosophila, rodent, primate and human. It emerges to develop the precision-nutrition therapeutics to slow age-related biological processes and treat diverse diseases. However, the nutritive advantages frequently diversify among individuals as well as organs and tissues, which brings challenges in this field. In this review, we summarize the different forms of dietary interventions extensively prescribed for healthspan improvement and disease treatment in pre-clinical or clinical. We discuss the nutrient-mediated mechanisms including metabolic regulators, nutritive metabolism pathways, epigenetic mechanisms and circadian clocks. Comparably, we describe diet-responsive effectors by which dietary interventions influence the endocrinic, immunological, microbial and neural states responsible for improving health and preventing multiple diseases in humans. Furthermore, we expatiate diverse patterns of dietotheroapies, including different fasting, calorie-restricted diet, ketogenic diet, high-fibre diet, plants-based diet, protein restriction diet or diet with specific reduction in amino acids or microelements, potentially affecting the health and morbid states. Altogether, we emphasize the profound nutritional therapy, and highlight the crosstalk among explored mechanisms and critical factors to develop individualized therapeutic approaches and predictors.
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Methionine restriction (MR) extends lifespan and improves several markers of health in rodents. However, the proximate mechanisms of MR on these physiological benefits have not been fully elucidated. The essential amino acid methionine plays numerous biological roles and limiting its availability in the diet directly modulates methionine metabolism. There is growing evidence that redox regulation of methionine has regulatory control on some aspects of cellular function but interactions with MR remain largely unexplored. We tested the functional role of the ubiquitously expressed methionine repair enzyme methionine sulfoxide reductase A (MsrA) on the metabolic benefits of MR in mice. MsrA catalytically reduces both free and protein-bound oxidized methionine, thus playing a key role in its redox state. We tested the extent to which MsrA is required for metabolic effects of MR in adult mice using mice lacking MsrA. As expected, MR in control mice reduced body weight, altered body composition, and improved glucose metabolism. Interestingly, lack of MsrA did not impair the metabolic effects of MR on these outcomes. Moreover, females had blunted MR responses regardless of MsrA status compared to males. Overall, our data suggests that MsrA is not required for the metabolic benefits of MR in adult mice.
Redox metabolism is crucial in host defense. Previously, it was shown that Borrelia burgdorferi induces the antioxidative metabolism in primary human monocytes. In this study, we explore how B. burgdorferi affects the anti-oxidative arm of redox metabolism, i.e. the generation of reactive oxygen species (ROS). Peripheral blood mononuclear cells (PBMCs) were exposed to B. burgdorferi and generation of ROS was determined both after acute stimulation and after re-stimulation with a secondary stimulus. Though the spirochete induces very low levels of ROS itself, it dramatically decreases the long-term capacity of PBMCs to generate ROS in response to serum-opsonized zymosan (SOZ). This was followed by a compensatory overshoot in ROS generation at later time points. The PI3K/Akt pathway and intracellular levels of methionine play an important regulatory role in this process. Dysregulation of oxidative metabolism may be a novel mechanism by which the spirochete modulates the human immune system and evades killing.
The prevalence of obesity is a worldwide phenomenon in all age groups and is associated with aging-related diseases such as type 2 diabetes, as well metabolic and cardiovascular diseases. The use of dietary restriction (DR) while avoiding malnutrition has many profound beneficial effects on aging and metabolic health, and dietary protein or specific amino acid (AA) restrictions, rather than overall calorie intake, are considered to play key roles in the effects of DR on host health. Whereas comprehensive reviews of the underlying mechanisms are limited, protein restriction and methionine (Met) restriction improve metabolic health and aging-related neurodegenerative diseases, and may be associated with FGF21, mTOR and autophagy, improved mitochondrial function and oxidative stress. Circulating branched-chain amino acids (BCAAs) are inversely correlated with metabolic health, and BCAAs and leucine restriction promote metabolic homeostasis in rodents. Although tryptophan (Trp) restriction extends the lifespan of rodents, the Trp-restricted diet is reported to increase inflammation in aged mice, while severe Trp restriction has side effects such as anorexia. Furthermore, inadequate protein intake in the elderly increases the risk of muscle-centric health. Therefore, the restriction of specific AAs may be an effective and executable dietary manipulation for metabolic and aging-related health in humans, which warrants further investigation to elucidate the underlying mechanisms.
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: Aging is a progressive and universal process originating endogenously that manifests during postmaturational life. Available comparative evidence supporting the mitochondrial free radical theory of aging consistently indicates that two basic molecular traits are associated with the rate of aging and thus with the maximum life span: the presence of low rates of mitochondrial oxygen radical production and low degrees of fatty acid unsaturation of cellular membranes in postmitotic tissues of long-lived homeothermic vertebrates in relation to those of short-lived ones. Recent research shows that steady-state levels of free radical-derived damage to mitochondrial DNA (mtDNA) and, in some cases, to proteins are lower in long- than in short-lived animals. Thus, nonenzymatic oxidative modification of tissue macromolecules is related to the rate of aging. The low degree of fatty acid unsaturation in biomembranes of long-lived animals may confer advantage by decreasing their sensitivity to lipid peroxidation. Furthermore, this may prevent lipoxidation-derived damage to other macromolecules. Taking into account the fatty acid distribution pattern, the origin of the low degree of membrane unsaturation in long-lived species seems to be the presence of species-specific desaturation pathways that determine membrane composition while an appropriate environment for membrane function is maintained. Mechanisms that prevent or decrease the generation of endogenous damage during the evolution of long-lived animals seem to be more important than trying to intercept those damaging agents or repairing the damage already inflicted. Here, the physiological meaning of these findings and the effects of experimental manipulations such as dietary stress, caloric restriction, and endocrine control in relation to aging and longevity are discussed.
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Oxidation of proteins by reactive oxygen species is associated with aging, oxidative stress, and many diseases. Although free and protein-bound methionine residues are particularly sensitive to oxidation to methionine sulfoxide derivatives, these oxidations are readily repaired by the action of methionine sulfoxide reductase (MsrA). To gain a better understanding of the biological roles of MsrA in metabolism, we have created a strain of mouse that lacks the MsrA gene. Compared with the wild type, this mutant: (i) exhibits enhanced sensitivity to oxidative stress (exposure to 100% oxygen); (ii) has a shorter lifespan under both normal and hyperoxic conditions; (iii) develops an atypical (tip-toe) walking pattern after 6 months of age; (iv) accumulates higher tissue levels of oxidized protein (carbonyl derivatives) under oxidative stress; and (v) is less able to up-regulate expression of thioredoxin reductase under oxidative stress. It thus seems that MsrA may play an important role in aging and neurological disorders.
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Little is known about the biochemical mechanisms responsible for the biological aging process. Our previous results and those of others suggest that one possible mechanism is based on the loss of glutathione (GSH), a multifunctional tripeptide present in high concentrations in nearly all living cells. The recent finding that life-long dietary restriction of the GSH precursor methionine (Met) resulted in increased longevity in rats led us to hypothesize that adaptive changes in Met and GSH metabolism had occurred, leading to enhanced GSH status. To test this, blood and tissue GSH levels were measured at different ages throughout the life span in F344 rats on control or Met-restricted diets. Met restriction resulted in a 42% increase in mean and 44% increase in maximum life span, and in 43% lower body weight compared to controls (P < 0.001). Increases in blood GSH levels of 81% and 164% were observed in mature and old Met-restricted animals, respectively (P < 0.001). Liver was apparently the source for this increase as hepatic GSH levels decreased to 40% of controls. Except for a 25% decrease in kidney, GSH was unchanged in other tissues. All changes in GSH occurred as early as 2 months after the start of the diet. Altogether, these results suggest that dramatic adaptations in sulfur amino acid metabolism occur as a result of chronic Met restriction, leading to increases in blood GSH levels and conservation of tissue GSH during aging.
The present investigation studies the effect of aging, short-term and long-term caloric restriction on four different markers of oxidative, glycoxidative or lipoxidative damage to heart mitochondrial proteins: protein carbonyls (measured by ELISA); N epsilon -(carboxyethyl)lysine (CEL), N epsilon -(carboxymethyl)lysine (CML), and N epsilon -(malondialdehyde)lysine (MDA-lys) measured by gas chromatography/mass spectrometry. Aging increased the steady state level of CML in rat heart mitochondria without changing the levels of the other three markers of protein damage. Short-term caloric restriction (six weeks) did not change any of the parameters measured. However, long-term (one year) caloric restriction decreased CEL and MDA-lys in heart mitochondria and did not change protein carbonyls and CML levels. The decrease in MDA-lys was not due to changes in the sensitivity of mitochondrial lipids to peroxidation since the measurements of the fatty acid composition showed that the total number of fatty acid double bonds was not changed by caloric restriction. The decrease in CEL and MDA-lys in caloric restriction agrees with the previously and consistently described finding that caloric restriction agrees with the previously and consistently described finding that caloric restriction lowers the rate of generation of reactive oxygen species (ROS) in rodent heart mitochondria, although in the case of CEL a caloric restriction-induced lowering of glycaemia can also be involved. The CEL and MDA-lys results support the notion that caloric restriction decreases oxidative stress-derived damage to heart mitochondrial proteins.
Groups of inbred A/J and C57BL/6J mice and hybrid F1 mice were fed low dietary protein (4% casein) or normal dietary protein (26% casein), with 50 mice in each of the six subgroups. For individual mice within subgroups, growth rate was negatively related to longevity; i.e., the slower the rate of growth the greater the life-span. Between subgroups, the longer the mean growth duration, the longer the mean lifespan. Positive relationships were obtained within subgroups for peak body weight and longevity. It is concluded that, for mice, slowing the rate of growth and increasing growth duration results in a significant increase in the life-span, and the life-span increment is not related to high peak body weight since high body weight, per se, was not correlated with life-span.
To test the hypothesis that reduced protein synthesis may increase life span by retarding genetic informational transfer during early life and reducing the use of the genetic code and thereby minimizing genetic imperfections as they may occur during late life, two approaches were used. In the first protein synthesis was depressed by the administration of cycloheximide, in the second by reducing the dietary protein level. One-day-old chick embryos were injected with either 0.8 gamma or 1.0 gamma of cycloheximide. On the second and third day of incubation both stage of development and heart rate were lower in the treated embryos. Growth was retarded throughout the 17 days of incubation as measured by size and DNA contents. As estimated by the activities of various enzymes per unit DNA, cells of the treated embryos were the same as normal ones of the same age. Sixteen-month-old female Wistar rats which had been previously maintained on a commercial diet (23.4% protein) were fed diets which contained either 24, 12, 8 or 4% casein throughout their remaining life span. Except for a lowering of the body weights of the animals fed the 4% casein diet, the body weights of the remaining animals were unchanged. Reducing the dietary protein level from 24% to 12% increased the life span (25%) of the animals.
The 50% mortality of female C57BL/6J mice fed ad libitum a diet which contained 26% or 4% casein, was 23.5 and 28 mo., respectively. Diet did not markedly affect the age-associated changes in the collagen content of the extractability of collagen of skin. In general, the activities of enzymes based on DNA were low in the restricted animals.
Carboxymethyllysine (CML) has been identified as a modified amino acid that accumulates with age in human lens proteins and collagen. CML may be formed by oxidation of fructoselysine (FL), the Amadori adduct formed on nonenzymatic glycosylation of lysine residues in protein, or by reaction of ascorbate with protein under autoxidizing conditions. We proposed that measurements of tissue and urinary CML may be useful as indices of oxidative stress or damage to proteins in vivo. To determine the extent to which oxidation of nonenzymatically glycosylated proteins contributes to urinary CML, we measured the urinary concentrations of FL and CML in diabetic (n = 26) and control (n = 28) patients. The urinary concentration of FL correlated strongly with HbA1 measurements and was significantly higher in diabetic compared with control samples (9.2 +/- 6.5 and 4.0 +/- 2.8 micrograms/mg creatinine, respectively; P less than 0.0001). There was also a strong correlation between the concentrations of CML and FL in both diabetic and control urine (r = 0.67, P less than 0.0001) but only a weakly significant increase in the CML concentration in diabetic compared with control urine (1.2 +/- 0.5 and 1.0 +/- 0.3 micrograms/mg creatinine, respectively; P = 0.05). The molar ratio of CML to FL was significantly lower in diabetic compared with control patients (0.25 +/- 0.12 and 0.43 +/- 0.16, respectively; P less than 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)
Eighteen male Wistar rats weighing 230 g (9 wk old) were fed casein diets containing 10% protein (HC), 50% protein (HP) or 10% protein plus 2% DL-methionine (MET) for 2 yr. In HC rats, mean body weight was 570 g; the carcass contained 13.5% protein and 37% lipid. The HP-fed rats had a 100 g lower body weight than HC rats due solely to a smaller amount of body lipid. Liver urea concentration and kidney weight were higher in HP rats than in HC rats. The body weight of MET-fed rats was lower than the other two groups and body lipid was only 30% that of HC rats. Histologic examination showed a normal aspect of the thoracic aorta from HC rats, whereas in HP, moderate signs of vascular aging--thicker intima and media with hypertrophy of smooth muscular cells (smc) with collagen enrichment and diffuse fibrosis--were observed. Aortas from MET rats also exhibited thicker intima and media due to smc hypertrophy. Some smc presented degenerative aspects and necrosis; other smc were replaced by chondroid cells and foci of fibrosis, resulting in a loss of the distension capacity of the aorta. Such an advanced stage of vascular aging is not normally found in 2-yr-old rats.